基于ERS-自噬稳态失衡探讨糖尿病肾病“毒瘀阻络”病理机制及益气解毒活血通络法的多靶点干预作用

Diabetic nephropathy (DN) is a main culprit of end-stage renal disease (ESRD). So far, the mechanism of DN is unclear and the treatment measures are deficient. In our and other studies, endoplasmic reticulum stress (ERS) was confirmed play key role in physiopathology of DN. Past research results showed that moderate ERS maintained cell homeostasis, while excess ERS accelerated cell death. Recent studies revealed that autophagy, as an important responser, was induced by ERS. ERS-autophagy homeostasis may play protective role in various pathologic condition. But high glucose injuried kidney cell autophagy function. Combined to TCM mechanism of DN, we proposed a hypothesis about DN mechanism: high glucose makes kidney cells fail to start autophagy (Deficiency of Spleen Qi and Kidney Qi). Besides, high glucose induces intracellular metabolic disorder and UPR (heat, blood stasis and toxin formation). ERS is induced in this process. "ERS-autophagy" homeostasis is disturbed in kidney cells. Then the kidney cells fail to metabolize intracellular toxin. The increased apoptotic cells and extracellular matrix (Toxin and blood stasis blockade kidney collateral) accelerate DN. According to our past clinical and pharmacological studies, we suggests treat DN by "tonifying Spleen Qi, clearing heat and removing toxin, promoting blood circulation to remove meridian obstruction". The aim of this study is to explore the role of ERS-autophagy in DN and the related targets regulated by Huangqi, Huangqin and Dilong.

糖尿病肾病（DN）作为终末期肾病元凶之一，其发病机制仍不明确，且治疗棘手。我们前期研究表明，内质网应激（ERS）作为主要的细胞应激方式，参与了DN的发病。适度的ERS促进蛋白的正确折叠、降解，维持细胞稳态；过度ERS则启动相关死亡程序，促进细胞死亡。近年研究发现，自噬作为ERS的重要应答机制，协同ERS维持细胞稳定。但长期高糖刺激，肾脏细胞自噬应答低下。结合DN的中医病机，我们提出假说：长期高血糖→肾脏细胞自噬功能低下（脾肾气虚）；高血糖→肾脏细胞内代谢失衡，错误折叠蛋白增多、相关炎症蛋白表达上调（化热化瘀成毒）→激活ERS；ERS-自噬稳态失衡的肾脏细胞不足以清除胞内毒性物质→细胞功能失调，凋亡，细胞外基质增多（毒瘀阻络），最终形成DN。结合前期临床及药理研究，我们提出"健脾益气，清热解毒，活血通络"法治疗DN，并探讨黄芪、黄芩、地龙三药调节"ERS-自噬"稳态的可能靶点。

项目背景：糖尿病肾病是一种严重威胁人类健康的疾病，发病率高，机制复杂，早期临床特点主要为微量白蛋白尿和肾脏肥大，益气解毒活血通络法对糖尿病肾病的防治具有明显的临床疗效，探讨其对糖尿病肾病的保护作用机制具有重要意义。本课题旨在为糖尿病肾病的中医药防治提供新的证据，并寻找其作用靶点。研究内容：（1）将db/m和db/db小鼠分为三组，db/m组（正常组），db/db组（2型糖尿病肾病模型组），db/db+AS-IV组（黄芪甲苷治疗组），给予db/db+AS-IV组小鼠喂养含有AS-IV的饲料（1g AS-IV混到1kg饲料中），db/m和db/db组喂养正常饲料，共计喂养12周，检测各组小鼠的进食量、饮水量、尿量、粪便量，尿白蛋白排泄率，肾脏病理情况，血生化及肾脏相关信号通路的变化。（2）通过体外高糖培养NRK-52E细胞，并使用AS-IV干预，探讨AS-IV的肾脏保护作用与NLRP3炎症小体表达、NLRP3/Caspase-1信号通路的关系。（3）提取2型糖尿病肾病db/db小鼠的肾脏线粒体，观察AS-IV对肾脏线粒体质量调控网络的调节作用。重要结果及关键数据：（1）AS-IV可明显减少db/db小鼠的尿白蛋白排泄，改善其病理损伤，但AS-IV对ERS信号通路作用不明显，对Akt/mTOR、NF-κB、Erk1/2信号通路作用比较明显。（2）通过体外高糖培养NRK-52E细胞，并使用AS-IV干预，结果显示AS-IV的肾脏保护作用与抑制NLRP3炎症小体表达、下调NLRP3/Caspase-1信号通路有关。（3）通过提取肾组织线粒体观察AS-IV对肾脏线粒体的作用，结果表明其肾脏保护机制可能与恢复线粒体质量调控网络有关。科学意义：AS-IV可能通过抑制NLRP3炎症小体表达、下调NLRP3/Caspase-1信号通路，恢复线粒体质量调控网络来保护2型糖尿病肾脏。为中药防治糖尿病肾病提供了新的证据。